Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells

Durocher, Yves; Pham, Phuong Lan; St-Laurent, Gilles; Jacob, Danielle; Cass, Brian; Chahal, Parminder S.; Lau, Cara J.; Nalbantoglu, Joséphine; Kamen, Amine

doi:10.1016/j.jviromet.2007.03.014

Cited by 77 publications

(60 citation statements)

References 31 publications

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“…This observation suggests that the incorporation of perfusion culture techniques to the process may increase upstream yields further still. We chose to restrict our efforts to adherent HEK293 cultures for reasons of simplicity and convenience, but given the use of PEI transfection in the production of rAAV in suspension cultures (Durocher et al, 2007;Hildinger et al, 2007), the adaptation of our upstream process to bioreactors appears feasible. A major advantage of such an approach would be the ability to use the same upstream process for both preclinical and clinical vector manufacture, which is desirable from a regulatory standpoint.…”

Section: Discussionmentioning

confidence: 99%

“…However, calcium phosphate triple transfection has generally not been considered ideal for large-scale rAAV production because of a lack of compatibility with suspension culture systems. Polyethylenimine (PEI) has been used as a transfection reagent to produce AAV vectors for some time (Grieger et al, 2006;Reed et al, 2006), but some promising results using this reagent have demonstrated the production of rAAV2 vectors in mammalian cell suspension culture with unpurified yields of 1-3Â10 13 vector particles per liter, which are comparable to yields from attached-mode transfection systems (Durocher et al, 2007;Hildinger et al, 2007). The advantages of PEI-based transfection are that it can also be performed in serum-free medium without the need for the medium exchanges that are typically required with conventional calcium phosphate-mediated transfection (Durocher et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

et al. 2010

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Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV-adenovirus, AAV-herpesvirus, and AAVbaculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1Â10 14 genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

et al. 2010

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“…HEK-293 cells are extensively used for the production of various viral vectors, including adenovirus (Kamen and Henry, 2004), adenoassociated virus (Durocher et al, 2007), retrovirus (Ghani et al, 2006) and lentivirus (Broussau et al, 2008). Due to their human origin, their ability to grow in serum-free suspension culture and their high transfectability, the HEK-293 cells also constitute an attractive platform for the transient or stable expression of recombinant proteins requiring proper post-translational modifications (Durocher and Butler, 2009;Durocher et al, 2002;Zhang et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

Henry

Durocher

2011

Metabolic Engineering

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“…Large-scale transient transfection has already been used for the production of proteins and viral vectors to generate preclinical and clinical material in a timely manner [8][9][10][11]. However, these early generation processes are generally not well characterized due to the lack of detailed key process parameter monitoring and sub-optimal control of bioreactor culture conditions.…”

Section: Introductionmentioning

confidence: 99%

Monitoring lentiviral vector production kinetics using online permittivity measurements

Ansorge¹,

Lanthier²,

Transfiguracion³

et al. 2011

Biochemical Engineering Journal

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Lentiviral vectors (LVs) are effective delivery vehicles that have been successfully used in gene and cell therapy. LVs are most commonly produced via the transient transfection of several plasmid constructs in adherent cell cultures. Recently, we described an efficient and scalable LV production in serum-free suspension cultures. To further facilitate the translation of LV-based interventions to the clinic, the robustness of the production process needs to be ensured to ultimately achieve a specified quality and quantity of LV production lots. However, routine processes are largely empirical, and strategies to monitor LV production kinetics in real-time have not yet been described.In this work, in situ real-time permittivity measurements were assessed to document the production of LVs. Characteristic process phases that were closely associated with LV production kinetics were identified. The permittivity signal evolution was interpreted by exploiting various independent online and offline monitoring measurements. Cellular membrane properties and, to a lesser extent, cell size were the main factors contributing to the permittivity variations. It is concluded that the permittivityrelated parameters can be used for the detection of viral release, allowing real-time assessment of process performance. The technology should thus greatly facilitate process development and optimization.Crown

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Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells

Cited by 77 publications

References 31 publications

Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase

Monitoring lentiviral vector production kinetics using online permittivity measurements

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